The present invention relates generally to the fields of cellular immunology and cancer therapy. More specifically, it relates to the preparation of immunogenic alloactivated cell compositions and the use of H2 receptor antagonists to affect lymphocyte interaction.
Significant progress has been made at the frontier of human cancer therapy by the development of new immunogenic compositions. A promising development has been the use of alloactivated lymphocytes in recruiting the host""s participation in the elimination of tumor cells.
One example is the method of treating tumors described in International application WO 95/20649. Alloactivated lymphocytes are prepared by collecting peripheral blood mononuclear cells from a healthy, unrelated donor, and stimulated in a mixed lymphocyte reaction with leukocytes of a cancer patient. At about the time of peak cytokine secretion, the cultured cells are harvested, prepared for human administration, and implanted into the bed of the tumor. The implanted cells apparently stimulate a host versus graft rejection, which then refocuses on bystander tumor cells to palliate the disease condition.
Another example is cancer immunotherapy using tumor cells combined with mixed lymphocytes, described in detail in International application WO 9816238. In this technology, third-party donor cells that have been alloactivated in culture are mixed with inactivated cancer cells from the patient, and administered intramuscularly as a vaccine. The host immune system is actively recruited by the alloactivated cells, and generates a specific systemic response against tumor antigen components present in the composition.
Both these technologies involve preactivation of the implanted lymphocytes. Not all combinations of human responder and human stimulator cells result in an equally strong alloreaction. Accordingly, there is a need to develop techniques to enhance the strength of alloreaction in the preparative cultures of immunogenic cells.
Type 2 receptors for the vasoactive amine histidine (H2 receptors) are present on several different cell types. In areas of medical research unrelated to the inventions described above, H2 receptor antagonists such as cimetidine have been used to affect immune function.
Properties of subpopulations of T cells bearing histamine receptors was described in the mid 1970""s (e.g., Plaut et al., J. Clin. Invest. 55:856, 1975; reviewed in Gordon et al., Michigan Academician 12:281, 1980). Experiments with H1 and H2 agonists and antagonists indicated that histamine-induced activation of suppressor T cells and the production of soluble suppressor factors were mediated through H2 receptors (Damle et al., J. Clin Immunol. 1:241, 1981). Eisenthal et al. (Cancer Immunol. Immunother. 21:141, 1986) showed that cimetidine augmented proliferation induced by allogeneic cells. Giulivi et al. (Int. J. Immunopharmacol. 8:517, 1986) showed that the addition of cimetidine to cultures of peripheral blood mononuclear cells enhanced monocyte transformation, possibly by blocking H2 receptors of T suppressor cells. Gifford et al. (Surgery 103:184, 1988 showed that cell-mediated cytotoxicity (CMC) is also increased by cimetidine in normal and down-regulated mouse splenocytes, and reverses CMC suppression by suppressor cells in a dose-dependent manner.
Cimetidine has been administered in various combination therapies to reverse suppression of anti-tumor immunity. See, for example, Osband et al. (Lancet 335:994, 1990), Marshall et al. (J. Biol. Resp. Modifiers 8:70, 1989), Richtsmeier et al. (Ann. Otol. Rhinol. Laryngol. 96:569, 1987, Osband et al. (Lancet i:636, 1981), Graham et al. (Sem. Urol. 11:27, 1993), Gold et al. (Am. J. Hematol. 44:42, 1993), Gold et al. (J. Immunother. 13:213, 1993), Carpinito et al. (Surg. Forum 37:418, 1986.), and Lavin et al. (Transplant. Proc. 24:3059, 1992).
U.S. Pat. No. 5,192,537 outlines a method of treating renal cell carcinoma using activated mononuclear cells, renal tumor antigen and cimetidine. Immunoreactive cells are sensitized for an antigenic marker associated with a malignant tumor in vitro. The activation involves collecting mononuclear cells from the patient, depleting suppressor T cells, suspending the mononuclear cells with autologous serum, and culturing the cells under conditions that immunize the cells against the tumor of the patient. The cells are then infused into the patient to treat the tumor. Optionally, in order to improve the efficacy of the cells, suppressor cells in the patient are inactivated by treating the patient with cimetidine.
U.S. Pat. No. 4,716,111 describes a process for producing human antibodies. Mononuclear cells are depleted of suppressor T cells bearing H2 receptors by binding to albumin-linked cimetidine. The remaining lymphocytes are then exposed to antigen, autologous serum, and a nonspecific lymphocyte activator. Examples proposed for activators are pokeweed mitogen, phytohemagglutinin, or supernatant from mixed lymphocyte cultures thought to contain allogenic effect factor.
U.S. Pat. No. 5,662,899 describes macrophages with enhanced cytotoxic activity by culturing in a medium containing a D3 vitamin and GM-CSF. Optionally, the medium used in the culturing of the macrophages also contains indomethacin or cimetidine.
International Patent Application WO 95/20649 relates to the ex vivo activation of immunoreactive cells. The proposed process involves contacting a first sample of mononuclear cells from a patient with OKT3 (anti-CD3 antibody) to produce an OKT3-derived culture supernatant, which is then used to activate a second cell sample. The suppressor cells in the mononuclear cell population may be functionally inactivated using cimetidine to inactivate suppressor cells and indomethacin to inactivate suppressor activity of monocytes.
European Patent Application EP 645147 outlines a method involving identifying and removing active lymph nodes in a patient having a tumor, and culturing the lymph node cells to obtain tumor-specific lymphocytes. Lymph node cells were expanded by incubating under various culture conditions. Biological response modifiers in the medium included IL-1, IL-2, IL-4, IFN-xcex3, OKT3 anti-CD3 antibody, indomethacin, and cimetidine (Example III). Calculated expansion indices indicated that cimetidine at 100 xcexcg/mL did not significantly alter expansion or phenotype at any time in the culture.
It has been discovered that adding H2 receptor antagonists to the medium of a preparative mixed lymphocyte culture increases the strength of the alloactivation within the first three days of the culture period. Extent of alloactivation at early culture times can be characterized by several functional criteria, including tetrazolium reduction, CD69 expression, intracellular esterase, and cytokine secretion. Enhanced cytokine secretion in turn is predicted to render the alloactivated cultures more effective for cancer treatment.
Accordingly, embodiments of the invention include a method for increasing the level of cytokine secretion, esterase activity, or CD69 expression by alloactivated lymphocytes, particularly during the first few days of alloactivation, comprising adding to the medium of an ex vivo culture of responder lymphocytes and allogeneic stimulator cells an H2 receptor antagonist.
Also embodied is a method of preparing a cultured cell population containing alloactivated human donor lymphocytes for treating a tumor or eliciting an anti-tumor immunological response in a human patient, comprising the steps of coculturing human lymphocytes allogeneic to the human patient with human leukocytes so as to alloactivate the lymphocytes, and harvesting the cocultured cells from culture at a time when the harvested cells, upon implantation in the bed of a solid tumor in the patient, are effective in the treatment of the tumor; wherein an H2 receptor antagonist is included in the culture medium. Compositions prepared according to this method are beneficially administered at or around the site of a solid tumor in a patient, with or without prior resection or partial resection of the solid tumor.
Also embodied is a method for preparing a cultured cell population containing alloactivated human donor lymphocytes for treating a tumor or eliciting an anti-tumor immune response in a human patient, comprising the steps of coculturing human lymphocytes allogeneic to the human patient with human leukocytes so as to alloactivate the lymphocytes, and combining the cocultured cells with tumor cells from the human patient or their progeny, progeny thereof, wherein an H2 receptor antagonist is included in the culture medium. Compositions prepared according to this method are beneficially administered in the form of an injectable cellular vaccine.
A non-limiting list of cancers suitable for treatment using compositions prepared according to these methods include melanoma, pancreatic cancer, liver cancer, colon cancer, prostate cancer, and breast cancer.